CH641445A5 - SIDE CHAIN FLUORINE VITAMIN D JOINTS. - Google Patents

Abstract

Fluorine-substituted vitamin D compounds, methods for preparation of such compounds and fluorinated intermediate compounds used in such methods are disclosed. The fluorine-substituted vitamin D compounds are characterized by vitamin D-like activity in stimulating intestinal calcium transport and bone mobilization and in promoting the calcification of rachitic bone.

Description

The invention relates to compounds with vitamin D-like activity. In particular, the invention relates to fluorine derivatives of vitamin D.

Vitamins D3 and D2 are well-known agents for the control of calcium and phosphorhormoostasis. These compounds stimulate intestinal calcium transport and bone calcium mobilization in normal animals or humans and are effective in preventing rickets. However, research over the past decade 40 has shown that vitamins D2 and D3 must be metabolized to their hydroxylated forms before biological activity is expressed. For example, recent evidence shows that 1,25-dihydroxy-vitamin D3, the dihydroxylated metabolite of vitamin D3, is the compound responsible for the biological effects mentioned above. In the same way, 1,25-di-hydroxy-vitamin D2 is the active form of vitamin D2.

State of the art

References to various vitamin D derivatives can be found in the patent literature and in other literature. See, for example, U.S. Patents 3,565,924, directed to 25-hydroxycholecalciferol; 3,697,559 directed to 1,25-dihydroxycholecalciferol; 3,741,996 directed to la-hydroxy-cholecalciferol; 3,743,661 directed to 5,6-tran, i-25-Hy-55 doxycholecalciferol; 3,907,843 directed to la-hydroxy-ergocalciferol; 3,715,374 directed to 24,25-dihydroxy-cholecalciferol; 3,739,001 directed to 25,26-dihydroxy-cholecalciferol; 3,786,062 directed to 22-dehydro-25-hydroxycholecalciferol; 3,847,955 directed to 1,24,25-tri-60 hydroxycholecalciferol; 3,906,014 directed to 3-deoxy-1 a-hydroxycholecalciferol; 4,069,321, directed to the production of various side chain fluorinated vitamin D3 'derivatives and side chain fluorinated dihydrotachysterin3 analogs.

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Presentation of the invention

It has also been found that fluorinated vitamin D compounds also have vitamin D-like activity.

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Such fluorine analogs are therefore useful compounds for the treatment of various diseases, such as osteomalacia, osteodystrophy and hyperparathyroidism.

In particular, the invention relates to fluorvitamin D compounds of the following general structure I and 5,6-fluorine D compounds of the following general structure II

R R

(I) and

(II),

R

R

where R in formula I is a steroid side chain of formula b) and in formula II a steroid side chain of formula a) or

(a)

or

(b)

where Rj is selected from the group of hydroxy, O-lower alkyl or O-acyl, wherein R2, R3, R4 and R5 are selected from the group of hydrogen, hydroxy, O-lower alkyl, O-acyl or Fluorine, with the proviso that at least one of R3, R4 and R5 must be fluorine, and wherein R6 represents hydrogen or lower alkyl. Best way to carry out the invention

These fluorine compounds and those compounds of formula I in which R represents a steroid side chain of formula a) are prepared by a process which involves treating hydroxylated 3,5-cyclovitamin D compounds with a fluorinating agent and obtaining the corresponding fluorine-3 directly , 5-cyclovitamin D Compound in which the fluorine is present on the carbon of the starting material originally occupied by the hydroxy function (s). These fluorocyclovitamin D intermediates are then solvolized in an aqueous or alcoholic solvent containing an acidic catalyst or in a solvent consisting of an organic carboxylic acid with a low

Molecular weight, to achieve both the 5,6-cis and the 5,6-trans-fluorvitamin D compounds of structure I or II, of which any acyl functions that may be present in the starting material or introduced during solvolysis by hydrolysis ( or reduction) can be removed to achieve the respective free hydroxy compounds.

Hydroxy-3,5-cyclovitamin D compounds of structure III below are used as starting materials for this fluorination process

R

ZO

R

used

where R is a steroid side chain of configuration

where R2, R3, R4 and R5 are selected from hydrogen, hydroxyl, O-lower alkyl and O-acyl, except that at least one of R3, R4 and Rs must be hydroxy, and wherein R6 is hydrogen or low Represents alkyl and wherein Z is lower alkyl.

In the present description and claims, the words "lower alkyl" denote a hydrocarbon radical containing 1 to 5 carbon atoms, which can be of normal or branched chain configuration (eg methyl, ethyl, isopropyl, butyl, isobutyl) and the word "acyl" denotes an aliphatic acyl group which contains 1 to 5 carbon atoms (e.g. acetyl, propionyl, butyryl) or an aromatic or substituted aromatic acyl group (e.g. benzoyl, nitrobenzoyl or chlorobenzoyl).

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The starting materials for fluorination, i.e. the 3,5-cyclovitamin D compounds of the general structure III can be prepared by known processes from 3-hydroxyvitamin D compounds which in turn are obtainable by synthesis or as isolated natural products (cf. for example Schnoes and DeLuca in Bioorganic Chemistry, EE Van Tamelen, ed., Vol. 2, Chapter 12, pages 299-335, Academic Press, NY 1978).

3,5-Cyclovitamin D compounds of general structure III can be prepared by the methods of Sheves and Mazur, J. Am. Chem. Soc. 97, 6249 (1975) and Paaren et al. Proc. Nat. Acad. Be. USA 75, 2080 (1978). The procedures given by these authors are general and the starting materials of structure III with any of the side chains given above are therefore readily available from the available 3-hydroxyvitamin D compounds.

Jones et al. (U.S. Patent 4,069,321) have described the preparation of various side chain fluorinated vitamin D3 derivatives and side chain fluorinated dihydrotachysterin 3 analogs. The manufacturing method recommended by these researchers involves fluorinating a precursor steroid and then converting the fluorsteroid into the desired fluorvitamin D compound.

However, it has now been found that side-chain and / or ring A-fluorinated vitamin D compounds or analogs of the above general structures I and II can be prepared much more expediently by directly introducing fluorine into hydroxy-cyclovitamin D compounds of the above general structure III by means of fluorination reagents, such as dialkylamino sulfur trifluoride, for example Diethylamino sulfur trifluoride. With such reagents, one or more fluorine substituents can be introduced into a vitamin D molecule by directly replacing one or more hydroxy functions in the starting material.

Middleton [J. Org. Chem. 40, 574, (1975)] has described the use of diethylaminosulfur trifluoride for the replacement of hydroxy functions by fluorine in organic compounds, but its applications are limited to simple and stable molecules which have no basis for evaluating the effectiveness of the reagent for the direct introduction of fluorine into vitamin D compounds without changing the sensitive double bond system. It is precisely this chemical reactivity of the vitamin C chromophore that has prompted other researchers in this field to take indirect and costly approaches when attempting to synthesize fluorvitamin D compounds. For example, Jones et al. (US Pat. No. 4,069,321) when recommending methods for the preparation of various fluorvitamin D analogs, diethylaminosulfur trifluoride only for the introduction of fluorine into precursor steroids which are subsequently converted into fluorvitamin D analogs. In fact, there is only a previous application of diethylaminosulfur trifluoride for the direct synthesis of 25-fluoro-vitamin D3 from 25-hydroxy-vitamin D3 [cf. Onisko, Schnoes and DeLuca, Tetrahedron Letters (No. 13) 1107 (1977)]. However, this one example gives no indication of the general utility of the fluorinating agent or of practical procedures for the preparation of other fluorvitamin D compounds.

It has now been found that the above diethylamino-sulfur trifluoride reagent can be used to introduce fluorine at various positions (e.g. carbon-24, 25, 26) of the side chain of a cyclovitamin D molecule, and to introduce fluorine to carbon 1 , and conveniently producing a variety of fluorvitamin

D compounds (structures I and II above), including those of the Jones et al. Patent (mentioned above), are effectively enabled.

Another advantage of the present process is the preparation of both 5,6-cis and 5,6-trans - fluorvitamin D compounds from the same cyclovita-min starting material and in the same process, thereby eliminating the need for separate synthesis of these two desired products becomes unnecessary. In addition, the use of 3,5-cyclovitamin D starting materials provides a highly useful temporary protection of the C-3 position and the triene system during fluorination. In the example of Onisko et al., Cited above, the C- 3-hydroxy group protected by the formation of a 3-0 - acyl function to avoid fluorination at C-3 as well as the rearrangement of the triene chromophore. We can confirm that fluorination of a vitamin D compound bearing an unprotected C-3 hydroxy group actually results in undesired products in which the trienchromophore is altered. The use of the stable 3,5-cyclovitamin D compounds of the general structure III as substrates for the fluorination not only avoids these difficulties, but also enables the desired regeneration of the desired C-3 substituent (C-3-hydroxy, O-acyl, O-alkyl) by subsequent acid-catalyzed solvolysis.

Finally, it was also found that more than one fluorine can be introduced at the same time simply by subjecting polyhydroxylated (e.g. di-, trihydroxy) cyclovitamin D starting materials to fluorination. Since the fluorination process involves the replacement of the free hydroxy function (s), it is also essential that any such functions in the starting material that are not to be replaced by fluorine are appropriately protected, e.g. by acylation, such as acetylene or benzoylate. Protection of hydroxyl groups can be conveniently achieved by known methods and, of course, after fluorination, the acyl groups can be easily removed, if desired, by hydrolysis under basic conditions.

The inherent protection of the C-3 position and the triene system and the simultaneous production of both cis and fra / is fluorvitamin products are the most notable features of the process according to the invention.

The general nature, scope and feasibility of the direct fluorination process by which fluorvitamin D compounds of general structure I above and fluorine-5,6-rra / 7.y-vitamin D compounds of general structure II above can be prepared from starting materials of general structure III, is illustrated in more detail by the following typical conversions.

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1. 25-hydroxy-6-methoxy-3,5-cyclovitamin D3 stage 1

► 25-Fluoro-6-methoxy-3,5-cyclovitamin D3

(Fluorine rank)

Level 2

> 25-fluorvitamin D3 + 25-fluoro-5,6- (solvolysis) -2rö / zs-vitamin D3

2. la-acetoxy-5-hydroxy-6-methoxy-3,5-cyclovitamin D3

| Step 1 (fluorination) 1 a-acetoxy-25-fluoro-6-methoxy-3,5-cyclovitamin D3

Stage 2 (solvolysis) la-acetoxy-25-fluorvitamin D3

, J, level 3 (hydrolysis)

la-hydroxy-25-fluorvitamin D3 +

la-acetoxy-25-fluoro-5,6-7rans-vitamin D3

| Stage 3 (hydrolysis) la-hydroxy-25-fluoro-5,6- (rans-vitamin D3

3. la, 25-dihydroxy-6-methoxy-3,5-cyclovitamin D3 stage 1

> l, 25-difluoro-6-methoxy-3,5-cyclo (fluorination) vitamin D3

Level 2

> 1,25-Difluorvitamin D3 + 1,25-Difluor- (Solvolysis) -5,6-trans-vitamin D3

4. 25-hydroxy-6-methoxy-3,5-cyclovitamin D2 stage 1

* 25-fluoro-6-methoxy-3,5-cyclovitamin D2 (fluorine rank)

Level 2

> 25-fluorvitamin D2 + 25-fluoro-5,6- (solvolysis) -trans-vitamin D2

5. 25,26-Dihydroxy-6-methoxy-3,5-cyclovitamin D3 Step 1

»25,26-difluoro-6-methoxy-3,5-cyclo- (fluorine rank) vitamin D3

Level 2

> 25,26-difluorvitamin D3 + 25,26-di- (solvolysis) fluoro-5,6-? Ra «y-vitamin D3

The cyclo-vitamin starting materials shown in reactions 1-5 above are prepared as described by Paaren et al. and Mazur et al. described in the references given above. In each of the illustrated examples, step 1 represents the fluorination reaction using diethylamino sulfur trifluoride and step 2 represents acid catalyzed solvolysis,

using any of the conditions of couples st al. in the above reference, and level 3 represents the hydrolysis of acyl protecting groups or of acyl groups introduced by solvolysis (if any).

It can be seen that the above reactions are intended to be illustrative only. The examples are presented to show that the process enables the convenient introduction of fluorine into both the ring A and the side chain of the steroid molecule, and in particular that vitamin D compounds and analogues are included

Fluorine substituents on any one or more of carbons 1, 24, 25 or 26 can be readily prepared from the corresponding hydroxylated cyclovitamin starting materials.

5 A preferred reagent for fluorination is diethylaminosulfur trifluoride (Middleton, J. Org. Chem. 40, 574 (1975)]. The reaction is conveniently carried out in a halogenated carbon solvent, such as methylene chloride, carbon tetrachloride or trichlorofluoromethane, at low temperature, for example —78 ° C. Short reaction times, for example 15-45 minutes, are suitable for the replacement of the hydroxy groups by fluorine. Although the reagent also attacks keto groups in the molecule, this takes place at a significantly lower rate, and therefore i5 protection of any existing keto function is normally not required under conditions where hydroxy groups are to be replaced.

As mentioned above, and illustrated by the above reactions, any hydroxy groups in the starting material that should not be replaced by fluorine in this process must be protected, e.g. acylated (acetylated, benzoylated). Acylation of hydroxygrapples is of course a well known process and is normally achieved by treating the hydroxy compound with an acylating agent (e.g. acetic anhydride, benzoyl chloride) in a suitable solvent (e.g. pyridine). Primary and secondary hydroxy groups in vitamin D compounds or their analogs are conveniently acylated using such re-3o agents and solvents at room temperature (or slightly elevated temperature, e.g. 50 ° C) for a period of 2-6 hours. The acylation of tertiary hydroxy groups naturally requires more severe conditions, e.g. elevated temperatures (e.g. 75-100 ° C) and suitable reaction times (e.g. 4-24 h.) It is preferred to carry out the reaction under a nitrogen atmosphere in order to avoid decomposition of material. The selective acylation of special hydroxyl groups can also be easily achieved. For example, l, 25-dihydroxy-6-methoxy-4o -3,5-cyclovitamin D31 acetate or l, 24.25-trihydroxy-6-methoxy-3.5-cyclovitamin D3 1,24-diacetate can be obtained by acetylation of l, 25-dihydroxy-6-methoxy-3,5-vita-min D3 or l, 24,25-trihydroxy-6-methoxy-3,5-cyclovitamin D3 at room temperature, because under such conditions 45 the tertiary hydroxy group not reacted. If selective acylation of chemically similar hydroxy groups is required, chromatographic separation of the products may be necessary. Thus, the Cl and C-24 monoacetates of 1,24,25-trihydroxy-6-methoxy-3,5-50-cyclovitamin D3 can be prepared by performing acetylation at room temperature and stopping the reaction before complete acetylation has occurred is. Under such circumstances, a mixture of four compounds is obtained. The non-acetylated starting material, the 1,24-diacetylated product, and the 1-acetoxy and 24-acetoxymonoacetylated compounds from which the desired monoacetates (as well as the 1,24-diacetate) are obtained by chromatography. Other partially acylated products which are required as starting materials for the subsequent fluorination are obtained in an analogous manner. Alternatively, partially acylated compounds can be obtained by first acylating all the hydroxy groups and then splitting off one or more of the acyl functions by hydrolysis. The 24,25-dihydroxy-6-methoxy-3,5-cyclo-vitamin D3 25-O-acyl compound can thus be obtained by partial hydrolysis of the 24,25-di-O-acyl derivative under basic conditions. These examples are called to

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show that many different methods are known and available for the production of O-protected vitamin D compounds, as may be required for the subsequent fluorination reaction.

Once the fluorine has been introduced into the molecule, all acyl protective groups can be removed in this step, if appropriate or expediently, by basic hydrolysis, e.g. by treating the acylated fluorocyclo-vitamin analog with 5% KOH in MeOH at a temperature of 25-80 ° C for 1-4 h. Alternatively, after the solvolysis of the cyclovitamin D intermediate to the ice and fraHs vitamin products, the de-acylation can be carried out as described below.

After the fluorine has been introduced, the fluorocyclo-vitamin derivatives are subjected to acid-catalyzed solvolysis using, for example, that of Sheves and Mazur and Paaren et al. in the conditions described above.

As shown by the above illustrative reactions, solvolysis leads to both the 5,6-cw-fluoro-vitamin D products and the corresponding 5,6 - / raws-fluoro-vitamin D analogs. These fluorinated eis and rans reaction products can conveniently be separated by chromatography at this stage (as described by Paaren et al. In the above reference) and can then be separately subjected to hydrolysis (if acyl protecting groups are to be removed) using Standard conditions, e.g.

0.1 KOH in methanol, 60 ° C, 1-4 h. Alternatively, the protecting groups can of course also be carried out before the separation of the eis and rans isomers.

Depending on the exact solvolysis conditions, 5,6-cis and rrans products with different C-3 substituents are obtained. Carrying out the solvolysis in a medium consisting of aqueous dioxane and a catalytic amount of acid (for example p-toluenesulfonic acid or methanesulfonic acid) leads to fluorinated 5,6-cis and -trans products which have a C- Wear 3-hydroxy substituents. Carrying out the solvolysis in an anhydrous alcoholic solvent (for example methanol, ethanol, isopropanol, etc.) which contains the acid catalysts mentioned leads to 5,6-cis and frans products which have a C-3-0-A1 -kyl substituents, wherein the alkyl group corresponds to the alkyl part of the alcohol solvent used. The solvolysis of fluorocyclovitamin D compounds in warm acetic acid (eg 50-60 ° C) leads to 5,6-cis and -frans-fluoro-vitamin D compounds which carry a C-3 acetoxy substitute and, if so Solvolysis of fluorocyclovitamins in dioxane / formic acid solvents is carried out, the corresponding 3-0-formyl-5,6-cis and -ra / i ^ products are obtained. The use of other organic acids (/C.B.trifluoroacetic acid) naturally leads to the analog 3-O-acyl products. If desired, these C-3-O-acylated products can easily be converted to the corresponding C-3-hydroxy compounds by mild base hydrolysis. From cyclovitamin D compounds of the general structure III, the preparation of 5,6-cis and -fraws-fluorvitamin D compounds according to the process according to the invention thus comprises an essential two-step sequence, namely

1. the direct introduction of fluorine, preferably using a dialkylamino sulfur trifluoride reagent, and

2. the subsequent solvolysis under acidic catalysis.

The previous hydroxy protection (e.g. through O-Acy-

lation) is only required in those cases where any of various available hydroxy groups in the starting material are to be replaced by fluorine. These

O-acyl protecting groups are easily removed if desired and, as indicated above, this step of deprotection can be carried out at any time after the fluorine has been introduced. For example, the removal of the O-protecting group immediately after the fluorination (before solvolysis) or immediately after solvolysis (before the separation of the ice and trans products) or finally after the separation of the ice and ZraMs isomers, that of the Solvolysis result, possible. A choice between these options would be based on convenience and / or the particular product desired.

example 1

la, 25-dihydroxy-6-methoxy-3,5-cyclovitamin D3 1-acetate l, 25-dihydroxy-6-methoxy-3,5-cyclovitamin D3 (5 mg), prepared according to the method of Paaren et al. (Proc. Nat. Acad. Sci. USA, 75, 2080 (1978)) is prepared with acetic anhydride (0.5 ml) and pyridine (2 ml) at 50 ° C under argon for 2 h. After the usual work-up, the 1-acetate product (5.5 mg) is isolated by high pressure liquid chromatography over a silica gel column, eluted with 2% 2-propanol / hexane.

Example 2

25-Fluoro-la-hydroxy-6-methoxy ~ 3,5-cyclovitamin D3 1-acetate l, 25-dihydroxy-6-methoxy-3,5-cyclovitamin D3 1-acetate (3 mg) in CH2C12 at - 78 ° C under argon is treated with diethyl amino sulfur trifluoride (5 mg). After a few minutes the reaction mixture can warm up to room temperature and is quenched with 5% K2C03. More CH2C12 is added, the organic phase is separated and the 25-fluorinated product is isolated by thin layer chromatography using a silica gel plate and 20% ethyl acetate in hexane as solvent to give 2 mg of the pure product.

Example 3

25-fluoro-la-hydroxy-6-methoxy-3,5-cyelovitamin D3

A sample of the 25-fluoro-cyclo-vitamin derivative obtained in Example 2 is dissolved in 0.1 M KOH / methanol solvent and heated to 50 ° C. for 2 hours. Water is then added and the hydrolyzed product is extracted into ether. Purification by thin layer chromatography on silica gel plates, developed with 30% ethyl acetate in Skellysolve B, gives pure 25-fluoro-1-hydroxy-6-methoxy-3,5-cyclovitamin D3.

Example 4

25-fluoro-la-hydroxyvitamin D3 and 25-fluoro-la-hydroxy-5,6-trans-vitamin D3

A solution of 3 mg of 25-fluoro-la-acetoxy-6-methoxy-3,5-cyclovitamin Ds (product of Example 2) in a 3: 1 mixture of dioxane and water (1.5 ml) containing 0 , 2 mg of p-toluenesulfonic acid is heated to 55 ° C. for 15 minutes. Saturated NaHC03 (2 ml) is then added, and the products are extracted into ether. The ether solvent is dried and evaporated and the residue is chromatographed on silica gel plates to separate 5,6-ice and 5,6-ans-25-fluoro-vitamin D products. Development with 25% ethyl acetate in Skellysolve B gives 1 mg of pure 25-fluoro-la-acetoxy-

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-vitamin D3 and 0.3 mg 25-fluoro-l'a-acetoxy-5,6-fran, r-vita-min D3. The hydrolysis of 25-fluoro-la-acetoxy-vitamin D3 in 0.1 m KOH / MeOH for 2 h at 50 ° C leads to a single product, 25-fluoro-lw-hydroxyvitamin D3: UV 265 nm ( e = 18 200); NMR (270 MHz) 0.55 (s, 18-CHS), 0.94 (d, J = 6 Hz, 21-CH,), 1.34 (d, J = 22 Hz, 26.27- (CH3 ) 2), 4.23 (m, 3a-H), 4.43 (m, lß-H), 5.33, 5.01 (2 m, 19-CH3), 5.85, 6.38 ( AB quartet, J = 11 Hz, 6 and 7-H's); Mass spectrum m / e 418.3197 (M +, 0.07, C27H4402F calculated 418.3222), 400.3141 (M + -H20, 0.22, calculated 400.3143), 382.3027 (M + -2 X H2 0, 0.23, calculated 382.3031), 380.3059 (M + -H20 -HF, 0.18, calculated 380.3069), 362.2974 (M + -2 X H20-HF, 0.18 calculated 362.2997) , 152.0842 (0.42, calculated 152.0840), 134.0736 (1.00 calculated 134.0734). The same hydrolysis of 25-fluoro-1 a-acetoxy-5,6-transvitamin D3 leads to 25-fluoro-la-hydroxy-5,6-frflas vitamin D3 in pure form (note × 270 nm); Mass spectrum, m / e 418.

Example 5

la, 25-difluoro-6-methoxy-3,5-cyclovitamin D3

A solution of 1 mg l, 25-dihydroxy-6-methoxy-3,5-cyclovitamin D3 (prepared by the method of Paaren et al. [Proc. Nat. Acad. Sci. USA, 75, 2080 (1978)] in CH2C12 at - 78 ° C under argon, it is treated with diethylamino-sulfur trifluoride (2 mg). After 10 minutes, the reaction mixture can warm up to room temperature and is quenched with 5% K2C03 2) l, 25-difluoro-6-meth-oxy-3,5-cyclovitamin D3 (250 | Xg) is isolated in pure form by high pressure liquid chromatography, using a silica gel column, eluted with 10% tetra-hydrofuran / Alternatively, the product can be purified by thin layer chromatography on silica gel using 20% ethyl acetate in Skellysolve B as a developing agent.

Example 6

la, 24 (R) 25-trihydroxy-6-methoxy-3,5-cyclovitamin D3 1,24-diacetate

A pyridine solution (0.2 ml) of 10 mg of 24 (R) 25-dihydroxyvitamin D3 is treated with 1.5 eq of p-toluenesulfonyl chloride at 0 ° C. for 48 h. The crude 3-tosyl product is obtained by dilution with saturated NaHC03 solution and extraction with ether. This product is treated in 2 ml of MeOH with 25 mg of NaHC03 and heated to 50 ° C. under N2 for 20 h. The cyclovitamin product is obtained by dilution with water and extraction with ether. Chromatography of the product on silica gel plates (40% ethyl acetate in Skellysolve B) gives 4 mg of 24 (R) 25-dihydroxy-6-methoxy-3,5-cyclovitamin D3 in pure form.

This product is obtained by treating with Se02 according to the procedure of Paaren et al. [Proc. Nat. Acad. Be. USA, 75, 2080 (1978)]: 4 mg of the cyclovitamine in CH2C12 solution (0.5 ml) are treated with Se02 (1 mg) and 10 (1L 70% t-butyl hydroperoxide for 30 minutes at 25 ° C. After the addition of NaOH solution (10%) the 1-hydroxy-cyclovitamin product is extracted into ether, evaporation of the solvent gives an oil which is chromatographed on silica gel plates using 50% ethyl acetate in Skellysolve B to give 1.5 mg la, 25 (R), 25-trihydroxy-6-methoxy - 3,5-cyclovitamin D3. This product is acetylated under usual conditions (0.2 ml acetic anhydride in 1 ml pyridine, 50 ° C, 2nd h). By diluting the reaction

mixed with water, extraction with ether and evaporation of the ether gives about 2 mg la, 24 (R), 25-trihydroxy - 6-methoxy-3,5-cyclovitamin D3 1,24-diacetate, sufficiently pure for the subsequent fluorination .

Example 7

25-fluoro-1,24 (R) -dihydroxy-6-methoxy-3,5-cyclovitamin D3 1,24-diacetate

The diacetoxy-cyclovitamin product obtained in Example 6 is fluorinated under conventional conditions (CH2C12 solvent, 3 mg diethylamino-sulfur trifluoride, -78 ° C, 15 min.). The reaction mixture can then warm to room temperature, is quenched with 5% K2C03 solution, and further CH2C12 is added. The organic phase is separated and the 25-fluorinated product is isolated and purified by thin layer chromatography on silica gel plates, using 25% ethyl acetate in Skellysolve B as the solvent system, to give 1 mg of 25-fluoro-la, 24 (R) - dihydroxy-5-methoxy-3,5-cyclovitamin D31,24-diacetate.

Example 8

25-fluoro-la, 24 (R) -dihydroxyvitamin D3 and 25-fluoro-l <a, 24 (R) -dihydroxy-5,6-tTans-vitamin D3

A dioxane solution of the 25-fluorocyclovitamin product as obtained in Example 7 is heated to 55 ° C. and treated with a 1: 1 mixture of dioxane: 98% formic acid (200 μl). After 15 minutes, ice-water is added and the products are extracted with ether. After evaporation of the solvent, the residue is taken up in 1: 1 dioxane / methanol (1 ml) and treated with 0.1 ml of an aqueous K2C03 solution for 5 minutes in order to hydrolyze the C-3 formate groups. Extracting the products in ether and chromatography on silica gel plates (using 40% ethyl acetate in Skellysolve B) gives 0.3 mg of 25-fluoro-la, 24 (R) -di-hydroxyvitamin D31,24-diacetate and 0.1 mg 25-fluoro-la, 24 (R) -dihydroxy-5,6-trans-vitamin D3 1,24-diacetate. The first-mentioned product is hydrolyzed (0, lm-KOH / MeOH, 50 ° C, 3 h) to 25 FIuor-la, 24 (R) -dihydroxyvitamin D3. The same hydrolysis of the 5,6-fra / js-diacetate leads to 25-fluoro-Ta, 24 (R) -dihydroxy-5,6-frans-vitamin D3.

Example 9

25-fluorvitamin D3 and 25-fluoro-5,6-tians-vitamin D3

A solution of 15 mg of 25-hydroxy-6-methoxy-3,5-cyclovitamin D3 [prepared by the method of Paaren et al. in proc. Nat. Acad. Be. USA, 75, 2080 (1978)] in 0.5 ml dichloromethane is added dropwise to a cooled (-78 ° C) mixture of diethylaminosulfur trifluoride (30 mg) in 0.5 ml CH2C12. After stirring for 5 minutes, the cooling bath is removed and the reaction mixture is warmed to room temperature. Aqueous NaHC03 (4%, 5 ml) and 10 ml CH2C12 are added. After separation of the organic phase, washing with H20, drying (Na ^ O ^ and evaporation of the solvent), the product, 25-fluoro-6-methoxy-3,5-cyclo-vitamin D3 on silica gel thin-layer plates, is developed with 10% ethyl acetate in Skellysolve B to give 10 mg of product This intermediate is solvolysed directly using the conditions of Paaren et al. (see reference above) [2.5 ml of a 3: 1 mixture of dioxane / Water containing 0.2 mg p-toluenesulfonic acid warmed to 55 ° C for 15 min, followed by addition of NaHC03 (3 ml) and Extra8

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the product in ether] to form a 3: 1 mixture (50% yield) of 25-fluorvitamin D3 and 25-fluoro-5,6-frarcs-vitamin D3. Separation on silica gel thin film using 15% ethyl acetate in Skellysolve B as solvent gives 25-fluorvitamin D3 as a colorless oil: UV (EtOH) Amax 265 nm; IR (CC14) 3620 (hydroxyl), 3080 cm-1 (exocyclic methylene); NMR (CDC13) 5 6.24 (d, J = 11 Hz), IH, C-6), 6.03 (d, J = 11 Hz, IH, C-7), 5.05 (m, IH, C-19), 4.82 (d, J = 2.6 Hz, 1H, C-19), 3.95 (t of t, ^ = 7.1 Hz, J2 = 3.6 Hz, IH, C -3), 1.34 (d, JHP = 21.3 Hz, 6H, C-26.27), 0.93 (d, J = 6 Hz, 3H, C-21), 0.54 (s, 3H, C-18); Mass spectrum m / e (relative intensity) 402.3284 (13, M +, 402.3298 calculated for C27H43OF), 360 (4), 271 (4), 253 (5), 136 (100), 118 (88), 61 (12), 59 (1); homogeneous with gas layer chromatography tR = 3.6 and 3.9 min. for the pyro- and isopyro-derivatives 2 mm X 6 '3% OV-lOl, 260 ° C oven, isothermal).

Example 10

25-Fluorvitamin Dz and 25-Fluor-5,6-tvä.ns-vitamin D2

A methylene chloride solution of 25-hydroxy-6-methoxy-3,5-cyclovitamin D2 [made from 25-hydroxyvitamin D2 according to the procedures of Paaren et al. Proc. Nat. Acad. Be. 75 2080 (1978)] is fluorinated and the product, 25-fluoro-6-methoxy-3,5-cyclovitamin D2, is isolated as described in Example 9. Solvolysis of this intermediate, exactly as described in Example 9, gives a (4: 1) mixture of 25-fluorvitamin D2 and 25-fluoro-5,6-rraj-vitamin D2, which is thin on silica gel -Layer plates (12% ethyl acetate / Skellysolve B as solvent) is separated, with the formation of pure 25-fluorvitamin D2 (UV Amax 265 nm; molecular weight = 414) and 25-fluoro-5,6-frarts vitamin D2 (UV Anm 270 nm; molecular weight = 414).

Example 11

25-fluoro-24 (RJ-hydroxyvitamin D3 and 25-fluoro-24 (R) - hydroxy-5,6-tva.ns-vitamin D3

The required 3,5-cyclovitamin D intermediate, 24 (R), 25-hydroxy-6-methoxy-3,5-cyclovitamin D3, is prepared from 24 (R), 25-dihydroxyvitamin D3, as in Example 6 described. This compound (5 mg) is acylated (0.3 ml acetic anhydride in 1.5 ml pyridine, at 50 ° C for 2 hours) to give the corresponding 24-monoacetate derivative (5 mg). This product is directly fluorinated and isolated, as described in Example 7, and the desired 25-fluorine product is then purified by thin layer chromatography (15% ethyl acetate in Skellysolve b). Solvolysis of this material, exactly as described in Example 8, gives the expected mixture of 3-formyloxy-24-acetoxy-25-fluorvitamin D3 and the corresponding 5,6- / rans isomer which is subjected to the hydrolysis (0.1 m-KOH-methanol, 1.5 h, room temperature) to remove formyl and acetyl groups. Evaporation of the solvent, addition of water and extraction with the ether gives 2.5 mg of crude products. High pressure liquid chromatography on a 0.7 X 25 cm column of 5 micron silica gel eluted with 2% isopropanol / hexane gives 25-fluoro-24 (R) -hydroxyvitamin D3 (1 mg): UV Amax 265 nm; NMR 5 0.55 (singlet, 18-methyl), 0.94 (doublet J = 5.9 Hz, 21-methyl), 1.34, 1.33

(two doublets, J = 22 Hz each, 26- and 27-methyl), 3.52 (multiplet, 24S proton), 3.95 (multiplet, 3a proton), 4.82, 5.06 (multiplets, 19 protons) 6.04, 6.24 (AB quartet, J = 12 Hz, 6.7 protons); Mass spectrum m / e (composition, m / e calculated) 418.3264 (C27H4302F, 418.3247), 398.3132 (C27H4202, 398.3185), 385.2904 (C26H38OF, 385.2906), 271.2050 (C19H270 , 271.2061), 253.1962 (C19H25, 253.1956), 136.0891 (C9H120, 136.0888), 118.0783 (C9H10, 118.0782); and 25-fluoro-24 (R) -hydroxy-5,6-trans-vitamin D3: UV Amax 272 nm; If the original solvolysis product mixture is subjected to hydrolysis with 0.1 M aqueous potassium carbonate, as described in Example 8, the 3-formyl group is removed, and the chromatography of the resulting mixture (silica gel plates, 20% ethyl acetate Skellysolve B as solvent) gives pure 24 (R) -acetoxy-25-fluorvitamin D3 and 24 (R) -acetoxy-25-fluoro-5,6-f / -anî-vitamin D3.

The new fluorine vitamin D compounds, prepared as described above, show considerable vitamin D-like biological effectiveness when examined in animals with a vitamin D deficiency. The vitamin D-like activities that these fluorvitamin D compounds exhibit include stimulating intestinal calcium transport, stimulating calcium mobilization from bone, and calcifying bone. To demonstrate these effects, a favorable test animal is the male infant rat, which is kept on a vitamin D deficient calcium diet or on a vitamin D deficient phosphorus diet, as described by Suda et al. J. Nutr. 100, 1049 (1970). In animals kept on a low calcium diet, intestinal calcium absorption can be controlled by the technique of the inverted intestinal sac by Martin and DeLuca [Am. J. Physiol. 216, 1351 (1969)], and bone calcium immobilization can be determined by the increase in serum calcium, as described, for example, by Blunt et al. Proc. Nat. Acad. Be. USA 61: 1503 (1968); the degree of endochondral calcification can be assessed using the line test method described in U.S. Pharmacopeia [15th revision, page 889, Mack Pubi. Easton, Pa. (1955)], using animals kept on the low phosphorus diet.

Using such methods, the biological effectiveness of the new fluorvitamin D compounds according to the invention can be conveniently demonstrated. A 5 fig dose of 25-fluorvitamin D2 administered to vitamin D deficiency, calcium deficiency leads to a considerable stimulation of intestinal calcium transport in animals and to an increase in serum calcium within 24 hours after administration.

Similarly, a 1 | Ag dose of la-hydroxy-25-fluorvitamin D2 is highly effective in stimulating intestinal calcium transport and bone calcium mobilization (measured by the increase in serum calcium levels) and the compound also promotes calcification of rachitic bone, measured according to the “line test” assessment.

Fluorine-5,6-fra / w-vitamin D compounds also show a pronounced vitamin D-like activity. For example, 25-fluoro-5,6-ira; w-vitamin D3 is effective in stimulating intestinal calcium transport, bone calcium mobilization and healing of rickets in these test animals.

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Claims (25)

641445 2. Compounds according to claim 1, wherein R4 is fluorine. 2nd PATENT CLAIMS 1. Compounds of Formula I R where R is a steroid side chain with the structures (I), V1 * 2 wherein R is a steroid side chain with the structure Rr is Rj is hydroxy, O-acyl or O-lower alkyl, R2 is hydrogen, hydroxy or O-acyl, R3, R4 and R5 are each selected from the group of hydrogen, hydroxy, O-acyl, O-lower alkyl and fluorine, with the proviso that at least one of R, Rs, R4 and R5 must be fluorine, and Is hydrogen or lower alkyl. 3rd 641445 where R is a steroid side chain with the configuration Rj is hydroxy, O-acyl or O-lower alkyl, R2, R3, R4 and R5 are each selected from the group of hydrogen, hydroxy, O-lower alkyl, O-acyl and fluorine, with the proviso that at least one of R3, R4 and R5 must be fluorine, and R6 is hydrogen or is lower alkyl, in which 3,5-cyclovitamin D compounds of the formula III is and R2, R3, R4 and R5 are each selected from the group consisting of hydrogen, hydroxyl, O-lower alkyl, O-acyl and Fluorine, with the proviso that at least one of R3, 5 R4 and Rg must be hydroxy and R6 is hydrogen or lower alkyl, treated with a fluorinating agent that produces fluorinated product, subjecting the obtained product to acid-catalyzed solvolysis, whereby a product is obtained that contains a mixture of fluorvitamin D and fluorine-5,6-trans-vitamin D products, separates these products chromatographically and each of them wins. 3. Compounds according to claim 1, wherein R3 is fluorine. 4. Compounds according to claim 2 or 3, wherein Rx is hydroxy. 5. Compounds according to claim 4, wherein R2 is hydrogen or hydroxy. 6. Compounds according to claim 1, wherein Re is methyl, with the stereochemical side chain configuration of ergosterol. 7. 25-fluorvitamin D2 according to claim 1. 8. la-hydroxy-25-fluorvitamin D2 according to claim 1. 9. Compounds of formula II Is 2o, <b> Rj is hydroxy, O-acyl or O-lower alkyl, R2 is hydrogen, hydroxy or O-acyl, R3, R4 and R5 are each selected from the group of hydrogen, hydroxy, O-acyl, O-lower alkyl and 25 fluorine, with the proviso that at least one of R3, R4 and R5 must be fluorine and hydrogen or low -Alkyl is. 10. Compounds according to claim 9, wherein R4 is fluorine. 11. Compounds according to claim 9, wherein R3 is fluorine. 3o 12. Compounds according to claim 10 or 11, wherein Rj Is hydroxy. 13. Compounds according to claim 12, wherein R2 is hydrogen or hydroxy. 14. Compounds according to claim 9, wherein R6 is methyl 35 with the stereochemical side chain configuration of Ergosterol. 15 starting material are present, against which fluorination is protected, by acylation before treatment with the fluorination reagent. 15. 25-fluoro-5,6-trans-vitamin D2 according to claim 9. 16. 25-fluoro-5,6-trans-vitamin D3 according to claim 9. 17. la-Hydroxy-25-fluoro-5,6-trans-vitamin Dz according to An-40 spoke 9. 18. 24-hydroxy-25-fluoro-5,6-trans-vitamin D3 according to claim 9. 19. la-hydroxy-25-fluoro-5,6-trans-vitamin D3 according to claim 9. 45 20. la, 24 (R) -dihydroxy-25-fluoro-5,6-trans-vitamin D3 according to claim 9. 20 of the solvolysis introduced acyl groups are removed by hydrolysis under basic conditions, either before or after the chromatographic separation of the solvolysis products. 21. Process for the preparation of fluorvitamin D compounds and fluorine-5,6-trans-vitamin D compounds of the formulas I and II so R R (II), 22. The method of claim 21, wherein one or more hydroxy groups present in the 3,5-cyclovitamin D 23. The method of claim 21 or 22, wherein one or more protective acyl groups or any during 24. The method of claim 21, wherein the fluorine reagent is a dialkylamino sulfur trifluoride. 25. The method of claim 24, wherein the fluorinating reagent is diethylamino sulfur trifluoride. (III), wherein Z is lower alkyl and R is a steroid side chain with the configuration (b) 3o Technical area